JP2009072144A - Method for saccharifying celluloses - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of efficiently promoting hydrolysis of celluloses by an enzyme while keeping lignin in a system without carrying out oxidative decomposition of lignin. <P>SOLUTION: The method for saccharifying the celluloses comprises saccharifying celluloses contained in biomass such as a non-edible part of farm products and ligneous unused material or waste material by using a hydrolase. The method for saccharifying the celluloses is characterized in that a substance having high affinity for lignin is previously added to celluloses and then, hydrolysis reaction of the celluloses is started by the hydrolase. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for saccharifying cellulose contained in biomass, and more particularly to a method for efficiently saccharifying celluloses by preventing a decrease in saccharification efficiency due to lignin.

  In recent years, development of technology for producing sugars such as glucose and xylose in particular has been promoted using celluloses, which are so-called woody and herbaceous biomass components such as wood chips, wood chips, rice straw and rice husks, as starting materials.

  Conventionally, in order to recover monosaccharides from woody and herbaceous biomass containing lignin, steaming or dilute sulfuric acid treatment is performed, and after collecting some hemicellulose, the cellulose component contained in the residue is hydrolyzed to produce sugar. A method for recovering (eg, glucose) is known.

  In the hydrolysis of cellulose, research and development is being promoted in the direction of using a hydrolase (eg, cellulase) rather than treatment with an acid or the like.

  Patent Document 1 discloses a method for saccharification of lignocellulose in which lignocellulose composed of lignin, hemicellulose, and cellulose is subjected to ozone treatment to decompose lignin in a previous stage using an enzyme.

Since this method has a low sugar recovery rate, the method of Patent Document 2 has been proposed to improve this. Patent Document 2 discloses a cellulose saccharification method in which hydrothermal treatment with hydrogen peroxide water is performed before enzymatic saccharification treatment to decompose lignin that is an obstacle to enzymatic saccharification and leaching from cellulose. .
JP 2006-141244 A JP 2007-74992 A JP, 2005-270056, A The fermentation inhibitory substance derived from lignin in the conversion process from sugar to alcohol is adsorbed with woody carbide.

  However, in the method described in Patent Document 2, since the treatment is performed by heating with hydrogen peroxide solution at 210 ° C. to 275 ° C. for about 30 seconds to 10 minutes, handling of the hydrogen peroxide solution is complicated in work. Moreover, there is a problem that it takes energy cost to perform the hot water treatment.

  As a result of studying a method for increasing the saccharification rate of celluloses by a method that does not incur work complexity and does not require energy costs, the present inventors have found the following findings. That is, in a system in which an enzyme and cellulose, hemicellulose, and lignin coexist, the enzyme has a high affinity for lignin, and therefore, based on the knowledge that cellulose hydrolysis by the enzyme is inhibited by lignin, lignin and When a reaction with a hydrolase was started after a substance having high affinity was added in advance, it was found that the reaction between the enzyme and celluloses was promoted, and the present invention was achieved.

  Therefore, an object of the present invention is to provide a method capable of efficiently promoting hydrolysis of celluloses by an enzyme without causing oxidative degradation of lignin while remaining in the system.

  The other subject of this invention becomes clear by the following description.

  The above problems are solved by the following inventions.

(Claim 1)
In a saccharification method of cellulose in which celluloses contained in biomass are saccharified using a hydrolase,
A method for saccharifying celluloses, comprising adding a substance having a high affinity for lignin in advance and then starting a reaction with a hydrolase.

(Claim 2)
The method for saccharifying celluloses according to claim 1, wherein the biomass is at least one selected from non-food parts of agricultural crops, wood-based unused materials or waste materials.

(Claim 3)
The method for saccharifying celluloses according to claim 1 or 2, wherein the substance having a high affinity for lignin is a protein.

(Claim 4)
The method for saccharifying celluloses according to any one of claims 1 to 3, wherein the substance having a high affinity for lignin is skim milk.

  ADVANTAGE OF THE INVENTION According to this invention, the method which can accelerate | stimulate the hydrolysis of celluloses with an enzyme efficiently can be provided, making it exist in a system, without oxidizing and decomposing lignin.

  Further, by masking lignin, the enzyme does not react with lignin, so that the enzyme can efficiently act on celluloses, and the amount of enzyme consumption can be minimized.

  Embodiments of the present invention will be described below.

  The present invention relates to a method for saccharification of celluloses in which saccharification of celluloses contained in herbaceous or woody biomass such as non-food parts of agricultural crops, wood-based unused materials or waste materials using a hydrolase is performed. It is characterized by having a step of adding a substance having a high affinity for lignin in the previous step of the step.

  In the present invention, the cellulose refers to either or both of cellulose and hemicellulose.

  FIG. 1 is a flowchart showing a biomass saccharification step in the present invention.

  1 is a steaming or acid treatment process. In this step, biomass such as non-food parts of agricultural crops or wood-based unused materials or waste materials is steamed or acid treated, and then filtered to separate the solution and solids (residues).

  The solid content (containing a large amount of cellulose) is preferably such that the size of the solid is 200 μm or less, and more preferably 100 μm or less. If the size of the solid material is larger than this, the hydrolase is less likely to act. After adjusting the solid content concentration with a buffer or the like, it is sent to step 2.

  2 is a step of adding a substance having a high affinity for lignin. Examples of the substance having a high affinity for lignin include proteins such as skim milk, lectin, gelatin, and preferably skim milk.

  Here, “affinity” can be measured by adsorption capacity. Hereinafter, a substance having a high affinity for lignin is simply referred to as a lignin adsorbing substance.

  The amount of lignin adsorbing substance added is preferably in the range of 0.1 to 30% by weight with respect to the solid content concentration of the residue.

  The lignin adsorbing substance of the present invention is adsorbed on lignin but not adsorbed on hydrolase or celluloses. For this reason, as mentioned above, there exists an effect which masks lignin, and the fall of the cellulose saccharification efficiency by lignin can be prevented.

  After the lignin adsorbing substance is added, the effect of lignin adsorption is enhanced by stirring well.

  Next, after masking the lignin with the lignin adsorbing substance, in the saccharification step 3, a saccharification enzyme such as cellulase is added according to the solid content concentration to saccharify the celluloses.

  The enzyme may be added in an amount corresponding to the cellulose in the residue.

  Since the lignin is masked, the enzyme does not adsorb to the lignin and the celluloses can be efficiently hydrolyzed, so that the amount of enzyme added more than necessary can be reduced.

  While maintaining the optimum pH and temperature of the enzyme to be used, the enzyme treatment is carried out for a certain period of time, followed by filtration in the filtration step 4, and divided into a liquid portion 5 in which the sugar is dissolved and a solid residue 6 to dissolve the sugar. The liquid part 5 is recovered. The recovered sugar is used for ethanol fermentation or the like.

  Examples of the present invention will be described below, but the present invention is not limited to such examples.

Example 1
The effect of adding lignin adsorbing substances on the relationship between lignin content and saccharification efficiency was investigated.

  After mixing 1.5 g of reagent cellulose (ratio 50%), 0.75 g of reagent xylan (hemicellulose) (ratio 25%), and 0.3 g of reagent lignin (ratio 10%), 0 so that the pH becomes 5.0. While adjusting with 0.05 M sodium acetate, water was added so that the solid content concentration was 4 wt% to prepare a sample (simulated biomass liquid).

  The skim milk was added to the sample in an amount of 0.5% by weight based on the solid content and stirred.

  Cellulase (Acremonium cellulase manufactured by Meiji Seika Co., Ltd.) loaded 40 FPU / g-DM, β-curcosidase (Genencore Kyowa Novozyme 188) loaded 5 IU (83.35 nkat) / g-DM, hemicellulase (manufactured by Yakult Pharmaceutical Co., Ltd., Cellulase Y) -NC) A load of 5 mg / g-DM was added and reacted at a temperature of 45 ° C. (water bath) at a stirring speed of 100 rpm for 48 hours.

<Sugar conversion efficiency>
After 48 hours, glucose analysis of glucose and xylose was performed by HPLC, quantification was performed by a standard method, and sugar conversion rates of cellulose and hemicellulose were calculated from the quantification results by the following formula.

<Solid liquefaction rate>
From the sample weight at the start of the test and the remaining sample weight after the test was completed, the solid content liquefaction rate was calculated by the following formula.

Example 2
An enzyme reaction was carried out in the same manner as in Example 1 except that the reagent lignin was changed to 0.15 g (ratio 5%), and the sugar conversion efficiency and the solid fraction liquefaction rate were calculated.

Comparative Example 1
The enzymatic reaction was carried out in the same manner as in Example 1 except that skim milk was not added, and the sugar conversion efficiency and the solid content liquefaction rate were calculated.

Comparative Example 2
The enzymatic reaction was carried out in the same manner as in Example 2 except that skim milk was not added, and the sugar conversion efficiency and the solid content liquefaction rate were calculated.

Reference Example An enzyme reaction was carried out in the same manner as in Example 1 except that the reagents lignin and skim milk were not added, and the sugar conversion efficiency and the solid content liquefaction rate were calculated.

  Table 1 shows the results of Examples 1 and 2, Comparative Examples 1 and 2, and Reference Example. Moreover, the graph which shows the relationship between sugar conversion efficiency and lignin content is shown in FIG.

  As the amount of lignin in the sample increased, the solid liquefaction rate and sugar conversion efficiency decreased. Among them, the sugar conversion efficiency decreases particularly as the amount of lignin increases (Comparative Examples 1 and 2), but Examples 1 and 2 to which skim milk is added have suppressed the decrease in the sugar conversion efficiency as compared with the Comparative Example. The sugar conversion efficiency is about twice as high as that of the comparative example.

Flow chart showing saccharification process of biomass in the present invention Graph showing the relationship between sugar conversion efficiency and lignin content

Explanation of symbols

1: Steaming or acid treatment step 2: Step of adding a substance having high affinity with lignin 3: Saccharification step 4: Filtration step 5: Liquid portion 6: Solid residue

Claims (4)

In a saccharification method of cellulose in which celluloses contained in biomass are saccharified using a hydrolase,
A method for saccharifying celluloses, comprising adding a substance having a high affinity for lignin in advance and then starting a reaction with a hydrolase.   The method for saccharifying celluloses according to claim 1, wherein the biomass is at least one selected from non-food parts of agricultural crops, wood-based unused materials or waste materials.   The method for saccharifying celluloses according to claim 1 or 2, wherein the substance having a high affinity for lignin is a protein.   The method for saccharifying celluloses according to any one of claims 1 to 3, wherein the substance having a high affinity for lignin is skim milk.

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